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WO1993021303A2 - Purification et expression ameliorees de proteines recombinees insolubles - Google Patents

Purification et expression ameliorees de proteines recombinees insolubles Download PDF

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Publication number
WO1993021303A2
WO1993021303A2 PCT/US1993/003652 US9303652W WO9321303A2 WO 1993021303 A2 WO1993021303 A2 WO 1993021303A2 US 9303652 W US9303652 W US 9303652W WO 9321303 A2 WO9321303 A2 WO 9321303A2
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Prior art keywords
host cell
protein
desired protein
insoluble
fraction
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PCT/US1993/003652
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English (en)
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WO1993021303A3 (fr
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Lawrence S. Cousens
Patricia Tekamp-Olson
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Chiron Corporation
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Publication of WO1993021303A2 publication Critical patent/WO1993021303A2/fr
Publication of WO1993021303A3 publication Critical patent/WO1993021303A3/fr

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Definitions

  • This invention relates to increasing intracellular expression of heterologous proteins in host cells and an improved method of purifying intracellularly expressed proteins from host cells.
  • protein products can be produced in large quantities.
  • One of the preferred methods of accomplishing this is to express the protein intracellularly. This may result in production of large quantities of insoluble, intracellular protein found in inclusion bodies, for example in the case of expression of fusion proteins.
  • fusion proteins may not be native in structure, they can sometimes be renatured or used "as is," e.g. for immunodiagnostics.
  • the major advantage of this approach is that it can yield very high levels of product.
  • Recombinant proteins produced as insoluble products in inclusion bodies have solubility properties that may interfere with their purification. These proteins generally require use of a denaturant, chaotrope or detergent for solubilization. In some cases, these treatments prevent the use of certain chromatographic procedures for purification, such as ion exchange. In addition, these treatments can solubilize non- proteinaceous contaminants that interfere with downstream processing, such as high molecular weight carbohydrates that may increase the viscosity of the solution.
  • the invention provides a separation method that purifies the desired insoluble protein based only on its solubility characteristics.
  • the method comprises: culturing a host cell in an effective amount of Cu ++ ; disrupting the host cell to produce a cell lysate; exposing the lysate to non-disulfide bond reducing or non-copper competing chaotropic conditions; separating the soluble contaminants from the insoluble pellet containing the desired protein; and incubating the pellet under disulfide bond reducing or copper competing chaotropic conditions.
  • Another object of the invention to provide a method for increasing insoluble protein expression.
  • the process comprises: providing host cell capable of expressing the desired protein; also providing an expression vector that does not contain any DNA encoding a functional metallothionein or a promoter regulated by Cu ++ concentration; and culturing the host cell in the presence of an amount of Cu ++ effective to increase expression.
  • a further object of the invention is an inclusion body comprising a protein aggregate obtained by the process comprising: culturing the host cell that expresses the desired protein in an effective amount of Cu ++ ; disrupting the host cell to produce a cell lysate; and separating said soluble fraction of the lysate from said insoluble fraction.
  • An “expression vector” comprises a coding sequence operably linked to any necessary control sequences, such as a promoter, terminator, and optionally a selectable marker. These control sequences are “operable” in a host cell if they enable the host cell to express the coding sequence.
  • An expression vector “free of functional metallothionein DNA” will not contain any DNA encoding a copper binding protein operable in the host.
  • a “promoter” is a DNA sequence that initiates and regulates the expression of a coding sequence when the promoter is operably linked to the coding sequence.
  • a promoter is "not regulated by Cu ++ concentration” if the promoter is not triggered or modulated by the presence or concentration of Cu ++ ions.
  • the bacterial lac promoter is triggered by the presence of lactose and is not regulated by Cu ++ concentration.
  • Metallothionein promoters are triggered by the presence of Cu ++ ions.
  • a “coding sequence” is a nucleic acid sequence that encodes a protein or peptide.
  • a “non-copper containing protein” does not specifically bind copper (e.g. insulin).
  • An example of a copper containing protein is superoxide dismutase, a protein that has a specific copper binding site.
  • Contaminants are any organic or inorganic matter (aside from solvents, and the like) that are not the desired protein.
  • Non-disulfide bond reducing chaotropic conditions are conditions that denature proteins but do not break disulfide bonds between cysteine residues. During this step, the desired protein remains insoluble. Such conditions are capable of solubilizing the contaminant proteins by either altering, for example, the state of hydration, the solvent environment, or the solvent-surface interaction. Examples of these conditions are effective amounts of urea, tetramethylurea, guanidine hydrochloride, sodium thiocyanate, and detergents, such as SDS.
  • An "effective amount of a non-disulfide reducing chaotropic agent” is the amount necessary to solubilize a substantial portion of the contaminants from the insoluble desired protein. These agents can be used alone or in combination.
  • the effective concentration will vary.
  • concentration of a non-disulfide reducing chaotropic agent e.g. SDS
  • concentration usually will not be less than 0.1 % , more usually the concentration will not be less than 0.25 % , preferably not less than 0.5%.
  • concentration will vary from protein to protein and host cell to host cell but may be determined empirically by routine experimentation.
  • Disulfide-bond reducing chaotropic conditions are conditions that denature proteins and break disulfide bonds. In addition to the above mechanisms, such conditions alter the solubility of proteins by breaking disulfide bonds that constrain the molecule. Examples of these conditions are effective amounts of any of the above non-disulfide bond reducing chaotropic agents in combination with a disulfide bond reducing agent such as: dithiothreitol, j8-mercaptoethanol, dithioerythritol, thioglycerol, tris(2-carboxyethyl)phosphine (TCEP) (Burns et al. J. Org. Chem.
  • TCEP tris(2-carboxyethyl)phosphine
  • the concentration of a non-disulfide reducing chaotropic agent (e.g. SDS) will be no greater than 10%; more typically, the concentration will be no greater than 7.5 % ; preferably, the concentration will be no greater than 5 % . Additionally, the concentration usually will not be less than 0.1%, more usually the concentration will not be less than 0.25%, preferably not be less than 0.5% .
  • an "effective amount of a disulfide-bond reducing agent” is an amount that breaks enough disulfide bonds to solubilize a substantial portion of the desired protein.
  • concentration of a disulfide reducing bond agent e.g. DTT
  • concentration usually will not be less than 1 mM; more usually the concentration will not be less than 5 mM, preferably not be less than 10 mM.
  • Optimal concentrations will vary from protein to protein and host cell to host cell but may be determined by routine experimentation.
  • Non-copper competing chaotropic conditions will not compete with the desired protein for copper by, for example, binding or reacting with the copper. Thus, the copper is not dissociated from the desired protein, which remains insoluble. Such conditions can solubilize contaminants by either altering, for example, the state of hydration, the solvent environment, or the solvent-surface interaction. Examples of these conditions are effective amounts of urea, tetramethylurea, guanidine hydrochloride, and detergents, such as SDS.
  • An "effective amount of a non-copper competing chaotropic agent” is the amount necessary to solubilize a substantial portion of contaminants while leaving the desired protein insoluble. These agents can be used alone or in combination.
  • the effective concentration will vary.
  • concentration of a non-copper competing chaotropic agent e.g. SDS
  • concentration usually will not less than 0.1 % , more usually the concentration will not be less than 0.25 % , preferably not be less than 0.5 % .
  • concentration will vary from protein to protein and host cell to host cell, but may be determined empirically by routine experimentation.
  • Copper competing chaotropic conditions compete or displace copper from the desired protein by binding or reacting with the metal ion. Under these conditions, the copper ion is dissociated from the desired protein, which is rendered soluble.
  • these conditions can also comprise a copper competing agent.
  • An "effective amount of copper competing agent” will be an amount that solubilizes a substantial portion of the desired protein.
  • An example of copper competing agents is DTT. Depending on the combination of agents the effective concentrations will vary. Typically, the concentration of a non- copper competing chaotropic agent (e.g.
  • SDS will be no greater than 10%; more typically, the concentration will be no greater than 7.5 % ; preferably, the concentration will be no greater than 5 % .
  • the concentration usually will not be less than 0.1 % , more usually the concentration will not be less than 0.25%, preferably not less than 0.5 % .
  • the concentration of copper competing agent e.g. DTT
  • DTT copper competing agent
  • the concentration usually will not be less than 1 mM; more usually the concentration will not be less than 5 mM, preferably not less than 10 mM.
  • Optimal concentrations will vary from protein to protein and host cell to host cell but may be determined by routine experimentation.
  • “Medium with enhanced aeration” is a solution of nutrients for the host cell enhanced with dissolved oxygen.
  • the oxygen can be added to the medium, for example, in the form of O 2 or a mixture of gases, such as air.
  • the amount of gas dissolved can be controlled by standard means, e.g., by adjusting the amount of gas bubbled into the medium, adjusting the stirring or shaking rate, adjusting the temperature of the medium, regulating the surface area of the volume of medium and the like. Agitating the medium can also increase the percentage of dissolved oxygen.
  • enhanced aeration means that the medium will contain a higher concentration of dissolved O 2 than a similar medium which is not agitated or otherwise aerated.
  • concentration of dissolved oxygen will be between 30% and 100% partial pressure, typically no greater than 60% partial pressure.
  • the optimal concentration will vary from protein to protein and from host cell to host cell but may be determined by routine experimentation.
  • an "effective amount of Cu ++,t is an amount sufficient to decrease the solubility of the desired protein, i.e. the protein will be resistant to solubilization under non-disulfide bond reducing or non-copper competing chaotropic conditions that solubilize most contaminants. The exact amount will vary depending on the protein and host cell, but can be determined empirically by examining the solubility properties of the expressed protein, e.g. on a SDS-PAGE gel.
  • Cu ++ can be added to the medium in the form of copper sulfate or copper chloride, for example. Typically, the concentration of Cu ++ will be no greater than 100 mM; more typically, the concentration will be no greater than 20 mM, preferably no greater than 5 mM.
  • the concentration usually will not be less than 10 ⁇ M; more usually the concentration will not be less than 100 ⁇ M, preferably not less than 500 ⁇ M.
  • the optimal concentration will vary depending on the desired protein, host cell, and medium but may be determined by routine experimentation.
  • An "amount of Cu ++ effective to increase expression" increases the amount of desired protein produced. The production of desired protein has increased if more protein is expressed when the host cells are cultured with than without Cu ++ .
  • the concentration of Cu ++ will be no greater than 100 mM; more typically, the concentration will be no greater than 20 mM, preferably no greater than 5 mM.
  • the concentration usually will not be less than 10 ⁇ M; more usually, the concentration will not be less than 100 ⁇ M; preferably not less than 500 ⁇ M.
  • the optimal concentration will vary depending on the desired protein, host cell, and medium but may determined by routine experimentation.
  • the term "HIN env protein” refers to an envelope protein from a human immunodeficiency virus, such as gpl20 and gp41 found in HIN-1 and HIN-2. "HIN env protein” includes both full-length proteins and antigenic fragments thereof (capable of immunoreactivity with anti-HIN antibodies).
  • Env2-3 is also an HIN env protein. The sequence is described by Luciw et al. Nature 312: 760-763 (1984). Env2-3 may include a fragment or epitope of the envelope protein. Preferably, it consists of amino acids 26 to 510 of gpl20.
  • IL-2 is either a portion or the complete interleukin-2 protein.
  • the amino acid sequence is described in U.S. Patent No. 4,518,584.
  • IL-2 will include a substantial portion of the complete protein.
  • IFN-jS refers to an interferon-0, such as IFN- 3,, T ⁇ l ⁇ - ⁇ 2 , and the like, or a fragment thereof. See U.S. Patent No. 4,518,584 for the amino acid sequence.
  • the terms refers to a substantial portion of the protein.
  • M-CSF refers to macrophage colony stimulating factor or a fragment thereof. Its amino acid sequence is described in U.S. Patent No. 4,929,700. Preferably, M-CSF includes amino acids 4 to 221 of the long clone with a mutation at position 59 from tyrosine to aspartate.
  • Fusion proteins expressed using the method of the invention include the following proteins: human Cu/Zn superoxide dismutase (SOD), human proinsulin, the putative Hepatitis C virus core (CORE), envelope (El), non-structural 3, 4, and 5 (NS3, NS4, & NS5) proteins.
  • SOD human Cu/Zn superoxide dismutase
  • CORE putative Hepatitis C virus core
  • El envelope
  • NS3, NS4, & NS5 proteins non-structural 3, 4, and 5
  • the sequences of the above proteins are known in the art.
  • the sequence of SOD is described in PCT WO85/01503.
  • the human proinsulin sequence is described in Bell et al. Nature 282: 525-527 (1979).
  • the entire amino acid sequence of HCV1 is described in EPO 388 232.
  • the fusion proteins below can include fragments of the proteins listed above, but generally will contain a substantial portion of the complete polypeptide sequence.
  • HCV1 is expressed naturally as a polyprotein. Any number of epitopes from the polyprotein can be useful as an immunodiagnostic. Epitopes from the following putative proteins particularly can be utilized: amino acids 1 to 192 (CORE); amino acids 191 to 384 (El); amino acids 1000 to 1500 (NS3); amino acids 1500 to 1960 (NS4 and amino acids 1960 to 3011 (NS5).
  • SOD/E1 refers to a fusion protein containing human Cu/Zn SOD sequence fused to a portion of the El region of HCV, preferably a fusion of the entire SOD amino acid sequence and amino acids 199 to 329 of HCVl from amino to carboxyl terminus of the fusion.
  • SOD/PI preferably is a protein that includes from the amino to carboxyl terminus the entire SOD protein linked to entire proinsulin gene by a linker with the amino acid sequence NSGPTPPSPGSPKR.
  • SOD/PI/E1 is a fusion protein that includes preferably the entire SOD gene, the entire proinsulin protein, a Lys-Arg-Ser-Asn-Ser-Thr linker, and amino acids 199 to 329 of HCVl, amino to carboxyl terminus.
  • C25 refers to a fusion of the entire SOD protein, which is linked at its carboxyl end to a portion of the NS3 region of HCV, preferably amino acids 1192 to 1931 of HCVl, which is linked to a portion of the core region of HCV, preferably amino acids 1 to 120 of HCVl.
  • SOD/NS5 refers to a protein that contains (amino to carboxyl) the entire SOD protein linked to a portion of the NS5 region of HCV, preferably amino acids 2054 to 2995 of HCVl .
  • Suitable proteins for the purification and expression methods will react with Cu ++ .
  • the desired protein may react with the Cu ++ by binding non-specifically with the metal.
  • the copper ion may bond with cysteine, methionine or histidine residues to reduce solubility.
  • Cu ++ may catalyze disulfide formation.
  • the instant invention may be more effective if additional cysteine, methionine or histidine residues, which may bind or react with Cu ++ , are incorporated into the desired protein.
  • the gene encoding the protein may be altered by in vitro mutagenesis to change residues to cysteine, for example.
  • This is done using a synthetic oligonucleotide primer complementary to a single-stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation.
  • the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage.
  • the new plaques will contain the phage having the mutated form as a single strand; 50% will have the original sequence.
  • the resulting plaques are hybridized with kinased synthetic primer under allele-specific conditions.
  • the empirical formula for calculating optimum tem- perature under standard conditions (0.9 M NaCl) is
  • T(°C) 4(N G + N c ) + 2(N A + N ⁇ ) - 5°C,
  • N G , N «, N . , and N ⁇ are the numbers of G, C, A, and T bases in the probe (J. Meinkoth et al, Anal Biochem (1984) 138:267-84). Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered.
  • a nucleic acid oligomer encoding a cysteine residue rich peptide can be added to the gene of the desired protein.
  • the oligomer can be added either to the 5' or 3' end of the desired protein gene.
  • the cysteine rich peptide may contain a cleavage site so that the peptide may be cleaved from the desired protein sequence. Examples of such sites are dibasic residues cleavable by KEX2 protease and Glu-Ala residues cleavable by dipeptidylaminopeptidase A.
  • These oligomers encoding the peptide may be produced by synthetic procedures, such as the triester method of Matteucci et al. (J.
  • nucleic acid oligomer After the nucleic acid oligomer is made, it can be ligated to the desired gene according to the procedures in Sambrook et al., "Molecular Cloning: A Laboratory Manual” (New York, Cold Spring Harbor Laboratory, 1989).
  • the desired protein gene sequence may be obtained either through publically available databanks, e.g. Genbank, or nucleic acid gene libraries. These sequences can be synthesized or cloned. Techniques for producing and probing nucleic acid sequence libraries are described, for example, in Sambrook et al., "Molecular Cloning: A Laboratory Manual” (New York, Cold Spring Harbor Laboratory, 1989). Synthetic oligonucleotides can also be used to generate the necessary gene. Insoluble aggregates of the desired protein can be formed from homologous proteins, as well as heterologous proteins. For example, altered conditions within the normal host can result in inclusion body formation of wild-type E. coli proteins.
  • the desired protein will be heterologous to the host cell and expressed via an expression vector.
  • the vector will comprise at least a promoter and terminator operably linked to the gene encoding the desired protein (and any modifications). These expression vector elements must be operable in the chosen host cell.
  • the expression vector will also contain a selectable marker and an origin of replication.
  • a promoter is a DNA sequence usually upstream or 5' of the gene to be expressed. This DNA sequence will initiate and regulate expression of the coding sequence. To initiate expression, promoter sequences are capable of binding RNA polymerase and initiating the downstream (3") transcription of a coding sequence (e.g. structural gene) into mRNA. A promoter may also have DNA sequences that can regulate the rate of expression by enhancing or specifically inducing or repressing transcription. These sequences can overlap promoter sequences that initiate expression. Most host cell systems possess regulatory promoter sequences. For example, a repressor protein in E. coli can bind the lac operator and thereby inhibit transcription of the 5' gene.
  • yeast alcohol dehydrogenase promoter which has an upstream activator sequence (UAS) that modulates expression in the absence of a readily available source of glucose.
  • UAS upstream activator sequence
  • viral enhancers not only amplify but also regulate expression in mammalian cells. These enhancers become active only in the presence of an inducer, such as a hormone or enzyme substrate (Sassone-Corsi and Borelli (1986) Trends Genet. 2:215: Maniatis et al. (1987) Science 236:1237).
  • Functional non-natural promoters may also be used, for example, synthetic promoters based on a consensus sequence of different promoters. Also, one may use promoters, which contain a regulatory region linked with a heterologous expression initiation region. Examples of hybrid promoters are the E. coli lac operator linked to the E. coli tac transcription activation region; the yeast alcohol dehydrogenase (ADH) regulatory sequence linked to the yeast glyceraldehyde-3-phosphate- dehydrogenase (GAP) transcription activation region (U.S. Patent Nos. 4,876,197 and 4,880,734); and the cytomegalovirus (CMV) enhancer linked to the SV40 (simian virus) promoter.
  • E. coli lac operator linked to the E. coli tac transcription activation region
  • yeast alcohol dehydrogenase (ADH) regulatory sequence linked to the yeast glyceraldehyde-3-phosphate- dehydrogenase (GAP) transcription activation region U.S
  • terminators are regulatory sequences located 3' of the stop codon of the coding sequences, such as polyadenylation and transcription termination sequences.
  • the terminator of native host cell proteins are operable when attached 3' of the desired protein's coding sequences. Examples are the Saccharomyces cerevisiae alpha-factor terminator and the baculovirus terminator.
  • viral terminators are also operable in certain host cells; for instance, the SV40 terminator is functional in Chinese Hamster Ovary cells.
  • selectable markers an origin of replication, and homologous host cells sequences may also optionally be included in an expression vector.
  • a selectable marker can be used to screen for host cells that potentially contain the expression vector. Such markers may render the host cell immune to drugs such as ampicillin, chloramphenicol, erythromycin, neomycin, and tetracycline. Also, markers may be b ⁇ osynthetic genes, such as those in the histidine, tryptophan, and leucine
  • An origin of replication may be needed for the expression vector to replicate in the host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the 2 ⁇ and autonomously replicating sequences, which are effective in yeast; the viral T-antigen, effective in COS-7 cells.
  • Expression vectors may integrate into the host cell genome or remain autonomous within the cell.
  • sequences homologous to sequences within the host cell genome must be provided.
  • the homologous sequences do not always need to be linked to the expression vector to be effective.
  • expression vectors can integrate into the Chinese Hamster Ovary cell (CHO) genome via an unattached dihydrofolate reductase gene.
  • CHO Chinese Hamster Ovary cell
  • yeast it is more advantageous if the homologous sequences flank the expression vector.
  • Particularly useful homologous yeast genome sequences are disclosed in PCT WO90/01800.
  • homologous baculovirus sequences are used to recombine with the virus that infects the cell. See Smith et al., Mol. Cell. Biol.
  • Bacterial hosts suitable for expressing a desired protein include, with limitation: Campylobacter, Bacillus, Escherichia,
  • Yeast hosts from the following genera may be utilized: Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia.
  • Immortalized mammalian host cells include but are not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines.
  • a number of insect cell hosts are also available for expression of heterologous proteins: Aedes aegypti , Autographa califomica. Bombyx mori. Drosophila melanogaster. and Spodoptera frugiperda (PCT WO 89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright
  • the transformation procedure to introduce the expression vector depends upon the host to be transformed.
  • Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and typically include either the transformation of bacteria treated with CaCl 2 or other agents, such as divalent cations and DMSO.
  • DNA can also be introduced into bacterial cells by electroporation or viral infection.
  • Transformation procedures usually vary with the bacterial species to be transformed. See e.g., (Masson et aL (1989) FEMS Microbiol. Lett. 60:273; Palva et aj. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. Nos. 036 259 and 063 953; PCT WO 84/04541, Bacillus), (Miller et aL (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter), (Cohen et al. (1973) Proc. Natl. Acad. Sci.
  • Transformation methods for yeast hosts are well-known in the art, and typically include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See e.g., (Kurtz et aL (1986) Mol. Cell. Biol. 6:142; Kunze et a (1985) J- Basic Microbiol. 25:141; Candida); (Gleeson et al- (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et aL Q986) Mol. Gen. Genet. 202:302: Hansenula); (Das et al. (1984) J. Bacteriol.
  • Methods for introducing heterologous polynucleotides into mammalian cells include viral infection, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al.. (1989). Bioessays 4:91.
  • the DNA sequence when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5' and 3' by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.
  • the newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1 % and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses.
  • the polyhedrin protein produced by the native virus at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 ⁇ in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope.
  • Cells infected with recombinant viruses lack occlusion bodies.
  • the transfection supernatant is plaqued onto a monolayer of insect cells by standard techniques. Typically, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies.
  • the medium to culture host cells will vary depending on the specific cell type chosen. Generally, the medium will contain all the nutrients needed to propagate the cells, e.g. amino acids, sugars, lipids, salts, vitamins, etc. Additional ingredients such as antibiotics, to limit growth of unwanted cells, or compounds like methotrexate, to enhance or induce expression, may also be included. Other factors, such as temperature, aeration, and pH, may need to be controlled and optimized. To aerate the medium, the gas mixture can be bubbled directly into the medium or supplemented to the exposed surface of the medium. Agitating the medium will help dissolve the gas into the media in both cases.
  • nutrients needed to propagate the cells e.g. amino acids, sugars, lipids, salts, vitamins, etc. Additional ingredients such as antibiotics, to limit growth of unwanted cells, or compounds like methotrexate, to enhance or induce expression, may also be included. Other factors, such as temperature, aeration, and pH, may need to be controlled and optimized.
  • the gas mixture can
  • Cu ++ ions can be added to the media in the form of copper salts, such as copper sulfate or copper chloride.
  • the copper ions can be present in the media throughout incubation of the host cells, but can be added immediately prior to protein expression. For instance, in E. coli expression, copper may be added when protein expression is induced with IPTG. Another important consideration is that the media components do not chelate all of the copper ions. For example, excess citrate, which binds copper, may reduce the effective Cu ++ concentration.
  • the medium is generally separated from the cells, usually by centrifugation, before the cells are disrupted.
  • sonicating or homogenizing the cell pellet for example, is effective to break the cells and produce a cell lysate.
  • certain host cells may be disrupted by enzymatic digestion of the cell wall or membrane. The digestion can be followed by osmotic shock, sonication, or detergent, if necessary. Lysozyme is commonly used for these enzymatic process. Autolysis and toluene are also effective for disrupting cells. Mammalian cells are easily disrupted by detergents or shearing. Preferably, the soluble fraction is separated from the cell lysate.
  • the insoluble material is pelleted by centrifugation, and the soluble fraction is removed.
  • filtering the cell lysate may be more economical.
  • Such filtering methods include ultrafiltration and diafiltration.
  • the insoluble fraction is resuspended and exposed to non-disulfide bond reducing or non-copper competing chaotropic conditions.
  • Homogenizers such as the OmmmixerTM, can easily resuspend the insoluble fraction, even if it is a solid pellet. Sonicating the fraction can also break the insoluble fraction into particles small enough to resuspend.
  • other components may be needed to optimize the purification.
  • a buffer will be added to maintain the pH of the solution.
  • the actual desired pH range is not fixed; however, the insoluble desired product must remain insoluble.
  • the pH may be adjusted within the desired range to aid in solubilizing contaminants. Salts, like NaCl, may also be advantageous, to better pellet the debris and possibly solubilize contaminants.
  • the resulting insoluble fraction must be separated from the soluble fraction. The same separation methods described above can be used.
  • the insoluble fraction is then resuspended in disulfide bond reducing or copper competing chaotropic conditions. Again, the insoluble fraction can be resuspended according to the above methods.
  • the disulfide reducing conditions may also optionally include a buffer, and salt in addition to the necessary components.
  • the desired protein is soluble, and the copper may still be present.
  • the copper can be removed by techniques known in the art (e.g. dialysis, desalting chromatography, diafiltration, or ultrafiltration).
  • the desired protein may also be renatured according to known procedures. See U.S. Patent No. 4,462,940.
  • the recovered proteins may be assayed for activity depending on the nature of the desired protein. For example, proteins expressed for use in immunoassays are assayed by quantifying their antigenicity using the appropriate antibodies.
  • Example 1 E. coli Expression of a Superoxide Dismutase/HCV Antigen Fusion
  • D1210(pCFlEF/C33c) is an E. coli strain transformed with an expression vector encoding a SOD/HCV antigen fusion protein.
  • This E. coli strain is a lac-repressor overproducing strain that carries the lacF and lacY + alleles. Its genotype is F, lacl + , lacO + , lacZ + , gal “ , pro " , leu “ , thi “ , end “ , hsm24 “ , hsr “ , recA “ , rpsL “ .
  • the vector contains an expression cassette with the tad promoter, the Shine Dalgarno sequences, a gene encoding human Cu/Zn superoxide dismutase (SOD), a peptide linker GIu-Phe-GIy, amino acids 1192 to 1457 of the HCVl isolate of Hepatitis C Virus, and a peptide tail Ala-GIu-Phe.
  • the expression vector also contains the ampicillin resistance gene and some pBR322 sequences. This transformed E. coli strain is described in E.P.O. Pub. Nos. 388 232 as pCFlEF/C33c, ATCC No. 67953.
  • the cell pellets were washed in TE (20 mM Tris buffer, pH 8.0 and 1 mM EDTA). The cells were disrupted by enzymatic digestion and sonication to produce cell lysates. Specifically, each cell pellet was resuspended in 15 ml TE containing 1 mg/ml of lysozyme and incubated for one hour at 4°C. The cells pellets were then sonicated with a Branson Sonifier 450 for 1-1.5 minutes at 70% power. The cell lysates were spun for five minutes at 15,000 rpm in a JA-20 rotor by a Beckman J2-21 centrifuge. The soluble fraction was decanted from the pelleted insoluble fraction.
  • TE 20 mM Tris buffer, pH 8.0 and 1 mM EDTA.
  • the cells were disrupted by enzymatic digestion and sonication to produce cell lysates. Specifically, each cell pellet was resuspended in 15 ml
  • the insoluble fractions were resuspended in 5 ml of a solution of non-copper competing, non-disulfide bond reducing chaotropic conditions (20 mM Tris, pH 8.0, 1 mM EDTA, 8 M urea).
  • the resuspended insoluble pellets were incubated in this solution for five minutes at room temperature.
  • the soluble fraction was separated by centrifuging the solution. Again, the soluble fraction was decanted from the insoluble pellet.
  • the resulting pellets were resuspended in 5 ml of a solution of disulfide bond reducing copper competing chaotropic conditions (20 mM Tris, pH 8.0, 1 mM EDTA, 8 M urea, and 50 mM DTT).
  • DG116(pLCSF221A) is an E. coli strain transformed with an expression vector encoding macrophage colony stimulating factor (M-CSF).
  • M-CSF macrophage colony stimulating factor
  • the vector contains an expression cassette with the pL promoter and a gene encoding amino acids 4 to 221 of the long clone of native M-CSF with a mutation at position 59 from a tyrosine to an aspartate.
  • the expression vector also contains the ampicillin resistance gene. An overnight culture was grown in 125 ml of L-broth with 10 mg/L of vitamin Bl and 50 mg/L of ampicillin.
  • the cell pellets were washed in TE (20 mM Tris buffer, pH 8.0 and 1 mM EDTA), and were disrupted by enzymatic digestion and sonication to produce cell lysates. Specifically, each cell pellet was resuspended in 15 ml TE containing 1 mg/ml of lysozyme and was incubated for one hour at 4°C. The cells pellets were then sonicated with a Branson Sonifier 450 1-1.5 minutes at 70% power. The cell lysates were centrifuged for ten minutes at 10,000 rpm in a JA-20 rotor by a Beckman 12-21 centrifuge. The soluble fraction was decanted from the pelleted insoluble fraction.
  • TE 20 mM Tris buffer, pH 8.0 and 1 mM EDTA
  • the insoluble fractions washed in 5 ml TE and resuspended in 5 ml of the same. One ml of each of the 5 ml suspensions was microfuged for ten minutes, and the soluble fraction was removed. The resulting pellets were resuspended in one ml of non-copper competing, non-disulfide bond - reducing chaotropic conditions (20 mM Tris, pH 8.0, 1 mM EDTA, 8 M urea). The resuspended insoluble pellets were incubated in this solution for ten minutes at room temperature. The soluble fractions were separated by microfuging at the same conditions.
  • the resulting pellets were resuspended in 1 ml of a solution of disulfide bond reducing, copper competing chaotropic conditions (20 mM Tris, pH 8.0, 1 mM EDTA, 8 M urea, and 50 mM DTT) and incubated at room temperature for ten minutes. The soluble fraction was separated by microfuging the solution.
  • a solution of disulfide bond reducing, copper competing chaotropic conditions (20 mM Tris, pH 8.0, 1 mM EDTA, 8 M urea, and 50 mM DTT
  • Example 3 Yeast Expression of an Superoxide Dismutase/Insulin Fusion
  • PO17(pYSI3) is a yeast strain transformed having an expression vector with a SOD/Insulin coding sequence.
  • the strain is 2150-2-3, whose genotype is (Mat a, ade 1. leu 2-04. cir°1.
  • the expression vector comprised: the ADH2/GAP hybrid promoter, SOD gene linked to the human proinsulin gene by DNA sequence encoding NSGPTPPSPGSPKR; and the ⁇ -factor terminator.
  • the expression vector also contains a leucine selectable marker and the 2 ⁇ sequences.
  • PO17(pYSI3) is described in U.S. patent application serial no. 07/680,046, which is the parent application to this application.
  • All buffers contained 1 mM PMSF and 1 ⁇ g/ml pepstatin.
  • the cell lysate was transferred to another tube, and the beads were washed twice with one packed cell volume (— 150 ⁇ l) of disruption buffer. The washes were pooled with the cell lysate. The cell lysate was split into three aliquots; each aliquot was centrifuged for ten minutes in a microfuge, and the soluble fraction was removed.
  • the remaining insoluble fractions were resuspended in approximately 200 ⁇ l of one of the following buffers; a) 50 mM sodium acetate, pH 3.5; b) 20 mM Tris-Cl, pH 8; or c) 50 mM glycine-OH, pH 10.5.
  • the resuspended fractions were centrifuged, and the soluble fraction was removed.
  • Each of the insoluble fractions was resuspended in a solution of non-disulfide bond reducing or copper competing chaotropic conditions (— 100 ⁇ l of 2 M urea in the appropriate (pH 3.5, 8, or 10.5) buffer) and incubated for 30 minutes at room temperature on a tilt-shaker.
  • each aliquot was split further into two portions (10 OD each) and centrifuged. The soluble fractions were removed.
  • each of the two pellets (the insoluble fractions) from the same pH extraction was resuspended in a 8 M urea in buffer of the appropriate pH with and without 20 mM DTT, a disulfide reducing, copper competing chaotropic agent. Samples were incubated for 30 minutes at room temperature on a tilt-shaker and centrifuged. The soluble fraction was removed from the centrifuged sample.
  • JSC308(pJS178) is a yeast strain transformed with an expression vector with sequences encoding a HIN-1 gp-120 antigen.
  • the yeast strain JSC308 was Mat A, ura3 ⁇ , leu 2-04, DM15, ci ⁇ ° and is described in EPO 340 986.
  • the vector contained an expression cassette with a ADH2/GAP hybrid promoter (U.S. Patent ⁇ os. 4,876,197 and 4,880,734), a D ⁇ A fragment encoding amino acids 26 to 510 from the env region of the HIN-SF2 isolate (Luciw et al. Nature 312: 760-763 (1984)), and the PYK terminator.
  • Also included on the vector were the ampicillin resistance gene, 2 ⁇ sequences, and the URA3 and LEU2 genes.
  • a culture of the transformed yeast strain was grown in leu " media with 5 % glucose at 30°C. The next day 0.5 ml of the overnight culture was transferred to 10 ml YEP media with 2% glucose and 0.5 mM CuSO 4 . The culture was incubated for 52 hours at 30°C in a 125 ml flask. The cells were harvested by centrifuging five ml of the culture at 3,000 rpm in a J6B rotor for ten minutes at 4°C in a Beckman J2-21 centrifuge. The cell pellet was washed with ten ml of 10 mM EDTA and centrifuged again. The washed cells were stored at -80 °C overnight.
  • the cell pellet was added to 0.5 ml of 20 mM Tris-Cl, pH 8.0 (lysis buffer) and 1 mM EDTA and 0.5 ml of 0.5 mm acid washed glass beads. The mixture was vortexed eight times for one minute intervals to produce a cell lysate. The total cell lysate was separated from the beads. Then, 0.5 ml of lysis buffer was added to the beads and vortexed briefly. The wash liquid was removed from the beads and pooled with the cell lysate. The pooled material was microfuged for ten minutes at 4°C. The supernatant (or soluble fraction) was removed, and saved for analysis.
  • the pellet was washed twice by resuspending the pellet by mixing with plastic rod and vortexing in 1 ml lysis buffer, followed by centrifugation in a microfuge. The supernatant (soluble fraction) was removed. The resulting pellet was resuspended in 1 ml lysis buffer. To test the solubility properties of env2-3, a lOO ⁇ l aliquot was centrifuged for 2 minutes in the microfuge at 4°C. The supernatant was discarded, and the pellet was resuspended in 200 ⁇ l lysis buffer supplemented with 1 % SDS (non- disulfide bond reducing, non-copper competing chaotropic conditions).
  • the solution was microfuged at room temperature. Then, the supernatant (soluble fraction) was removed, and the pellet was resuspended in 20 ⁇ l of lysis buffer supplemented with 1 % SDS and 50 mM DTT (disulfide bond reducing, copper competing chaotropic conditions. After incubating the solution at room temperature for 15 minutes, the solution was centrifuged for 10 minutes at room temperature.
  • lysis buffer supplemented with 1 % SDS and 50 mM DTT (disulfide bond reducing, copper competing chaotropic conditions.
  • the proteins solubilized by the different methods were analyzed by SDS gel electrophoresis. The results demonstrated a dramatic purification of the desired protein. Nearly all the contaminants were soluble in the non-disulfide bond reducing, non-copper competing chaotropic conditions and were removed by the process of the invention.
  • JSC308(pSOD/El) is a yeast strain transformed with an expression vector with sequences encoding SOD linked to the HCV El protein antigen.
  • the yeast strain JSC308 was Mat A, ura3 ⁇ , leu 2-04, DM15, cir° and is described in EP 340 986.
  • the vector contained an expression cassette with a ADH2/GAP hybrid promoter (U.S. Patent Nos. 4,876,197 and 4,880,734), a DNA fragment encoding amino acids 199 to 329 from the putative El protein of the HCVl isolate (EPO 388 232), and the ⁇ -factor terminator. Also included on the vector were the ampicillin resistance gene, 2 ⁇ sequences, and the LEU2 gene.
  • a culture of the transformed yeast strain was grown in leu " media with 5% glucose at 30°C. The next day 0.5 ml of the overnight culture was transferred to 10 ml YEP media with 2% glucose and 0.5 mM CuSO 4 . The culture was incubated for 52 hours at 30°C in a 125 ml flask. The cells were harvested by centrifuging five ml of the culture at 3,000 rpm in a J6B rotor for ten minutes at 4 °C in a Beckman J2-21 centrifuge. The cell pellet was washed with ten ml of 10 mM EDTA and centrifuged again. The washed cells were stored at -80°C overnight.
  • the cell pellet was added to 0.5 ml of 20 mM Tris-Cl, pH 8.0 (lysis buffer) and 1 mM EDTA and 0.5 ml of 0.5 mm acid washed glass beads. The mixture was vortexed eight times for one minute intervals to produce a cell lysate. The total cell lysates was separated from the beads. Then, 0.5 ml of lysis buffer was added to the beads and vortexed briefly. The wash liquid was removed from the beads and pooled with the cell lysate. The pooled material was microfuged for ten minutes at 4°C. The supernatant (or soluble fraction) was removed, and saved for analysis.
  • the pellet was washed twice by resuspending the pellet by mixing with plastic rod and vortexing in 1 ml lysis buffer, followed by centrifugation in a microfuge. The supernatant (soluble fraction) was removed. The resulting pellet was resuspended in 0.5 ml of water.

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Abstract

Un procédé d'isolement d'une protéine voulue consiste à produire une cellule hôte exprimant la protéine voulue sous la forme d'un agrégat insoluble, à mettre en culture la cellule hôte avec une dose efficace de Cu++, à désunir la cellule hôte pour produire un lysat, à mettre en incubation la fraction insoluble dans des conditions chaotropes non réductrices de ponts disulfure ou non concurrentes du cuivre afin de solubiliser les contaminants, à séparer la fraction insoluble de la fraction soluble, et à exposer la fraction insoluble à des conditions chaotropes réductrices de ponts disulfure ou concurrentes du cuivre afin de solubiliser la protéine voulue.
PCT/US1993/003652 1992-04-16 1993-04-16 Purification et expression ameliorees de proteines recombinees insolubles WO1993021303A2 (fr)

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WO1995020664A3 (fr) * 1994-01-28 1995-12-28 Abbott Lab Systemes d'expression mammaliens pour des genes d'enveloppe du virus de l'hepatite c
EP0696640A3 (fr) * 1994-08-12 1997-12-29 Boehringer Mannheim Gmbh Antigènes de recombinaison venant de la région NS3 du virus de l'hépatite C

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JP2004511753A (ja) 2000-05-04 2004-04-15 イエール　ユニバーシティー タンパク質活性のスクリーニング用タンパク質チップ
WO2003102013A2 (fr) 2001-02-23 2003-12-11 Gonzalez-Villasenor Lucia Iren Procedes et compositions de production de peptides recombinants
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WO2003011285A1 (fr) * 2001-08-01 2003-02-13 Merck & Co., Inc. Derives de la benzimidazo[4,5-f]isoquinolinone
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WO1995020664A3 (fr) * 1994-01-28 1995-12-28 Abbott Lab Systemes d'expression mammaliens pour des genes d'enveloppe du virus de l'hepatite c
US5610009A (en) * 1994-01-28 1997-03-11 Abbott Laboratories Mammalian expression systems for hepatitis C virus envelope genes
EP0696640A3 (fr) * 1994-08-12 1997-12-29 Boehringer Mannheim Gmbh Antigènes de recombinaison venant de la région NS3 du virus de l'hépatite C

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